1. Field of the Invention
The invention concerns specific nucleic acid probes and a method for the detection of the pathogen Neisseria gonorrhoeae.
2. Description of the Prior Art
N. gonorrhoeae is the pathogen of gonorrhoea which is still today the most frequent notifiable infectious disease in the world (World Health Statistics Annual (1979), Geneva, WHO). In men, the genital infection manifests itself as a purulent inflammation and swelling of the urethra. These symptoms occur in 90% of cases of infection. If left untreated, the infection can ascend and after several weeks produce symptoms of prostatitis. In contrast, in women no or only slight symptoms occur in 50% of cases of infection. The infection primarily affects the cervix, but also the urethra. In 10 to 15% of women the infection spreads to the fallopian tubes which can also lead, inter alia, to sterility. In 1 to 3% of the cases a systemic invasion by the pathogen can occur in men and women which can lead to arthritis, endocarditis and peritonitis. Since the course of the infections is often asymptomatic, many carriers contribute unknowingly to the spread of the disease (Davis et al., In: Microbiology; Harper International Edition).
The species Neisseria is a group of closely related gram-negative diplococci which includes pathogenic as well as non-pathogenic species. The differentiation between N. gonorrhoeae and other non-pathogenic species which colonize the mucous membranes of man is therefore very important for the subsequent treatment. There is thus often the risk of mistake if the diagnosis of gonorrhoea is based solely on a microscopic examination.
A definitive diagnostic test for Neisseria gonorrhoeae requires the preparation of a culture. In this connection, difficulties often occur in the transport of material and the culture because the gonococci, as a result of their autolytic enzyme systems, are extremely sensitive to environmental influences such as change in temperature and dehydration. In addition, the culture is tedious since the incubation period is at least 20 hours. Furthermore, the culture as well as the definitive identification of the pathogen can be difficult and must therefore be carried out in special microbiological laboratories. The cultured gonococci are differentiated by carbohydrate degradation, oxidase reaction, antigen detection by coagglutination and fluorescent antibody screening.
A selective medium is used to culture N. gonorrhoeae on which, however, non-pathogenic Neisseria species such as e.g. N. lactamica can also grow. For this reason there is a risk of mistake which can only be excluded by exact and time-consuming biochemical differentiation. Attempts have therefore already been made to develop diagnostic tests which enable the rapid and specific detection of Neisseria gonorrhoeae.
In recent years, the technique of nucleic acid hybridization, which is attractive from the point of view of speed, has been developed for the identification of pathogenic organisms. Attempts have also been made to use nucleic acid probes for diagnosis with respect to Neisseria gonorrhoeae.
Nucleic acid probes have to fulfil two criteria in order to be used diagnostically. They must be specific i.e. the probe should only hybridize to the nucleic acid of the pathogen to be detected in order to exclude "false positive" test results. They must also be sensitive, i.e. the detection of only a few pathogens should also be possible in order to exclude "false negative" test results during an early stage of the infection.
A few years ago it was already recognized that it could be possible to specifically detect organisms using nucleic acid probes which are complementary to ribosomal RNA (rRNA). Such probes have the advantage that they are very sensitive since between 1000 copies and 10000 copies (of the rRNA) are present in each cell.
The method of identifying organisms using such rRNA-specific probes has already been described several times (EP-B 0155359, WO 84/02721, EP-A 0076123).
Also in the case of Neisseria gonorrhoeae, probes, whose sequences are complementary to regions of the 16S rRNA, have been used for the specific detection of this pathogen. In this connection, the sequence of the probes was derived from 3 regions of the 1544 nucleotide long 16S rRNA from N. gonorrhoeae (nucleotide position 125-150, 455-485, 980-1015). The above-mentioned probes were hybridized as a mixture to the entire RNA which was isolated from different Neisseria species (N. gonorrhoeae, N. Meningitidis, N. cinerea, N. lactamica, N. mucosa and N. subflava) and Kingella kingae. The probes were regarded to be specific for N. gonorrhoeae (EP-A 0272009).
This mixture of probes was tested with a selection of different species (see below). The investigation showed that the known probes hybridized with the nucleic acid of Neisseria denitrificans, a non-pathogenic species which is, however, closely related to Neisseria gonorrhoeae.